Quinoxaline derivatives are an important class of benzoheterocycles which has received much attention in recent years owing to their both biological properties and pharmaceutical applications. These derivatives are particularly interesting since some of them showed anti-microbial [1-9], anticancer [10-25], antimalarial [26-31], antiinflammatory [32-33], antinociceptive [34-36], antitubercular [37-39], anthelmintic [40-41], antidiabetic [42] and antiepileptic [43-44] properties. There is no report on the synthesis of indeno[1, 2-b]quinoxalin-11-ylidenamines and hence our research has focused to synthesize new indeno[1, 2-b]quinoxalin-11-ones substituted by aromatic amine moieties in 11 position and to investigate their antinociceptive, anti-inflammatory and antiepileptic activities.

2. Materials and methods

2.1. Chemistry

Titled compounds were prepared as shown in Figure1. Melting points were determined by Veego melting point apparatus and are not corrected. Thin layer chromatographic analysis were performed on a Merck grade Aluminum foil GF254 plates of 0.25 mm thickness in chloroform: water system (9:1). Spots were visualized under UV light. Infrared spectra were obtained on a Perkin Elmer-1600 series FTIR spectrophotometer using potassium bromide discs. Nuclear magnetic resonance spectra were recorded on Brucker 400 MHz spectrophotometer. Chemical shifts are reported in parts per million (δ) units relative to internal standard tetramethylsi-lane. Mass spectra were recorded on Joel JMS-DX 303 mass spectrophotometer. Elemental analysis was performed on Heraceus Carlo Erba 1108 and the analysis indicated by the symbols of the elements was within ±0.4 % of theoretical values.

The animals used for the studies were in accordance with principles of laboratory animal care and were approved by Institutional animal ethical committee. The antinociceptive evaluation was carried out using acetic acid induced writhing method. Swiss strain albino mice of either sex weighing 25-30 g were used for this study. The antiinflammatory activity evaluation was carried out using carrageenan induced rat paw edema method. Albino rats of Wistar strain weighing 100-200 g of either sex were used for this study.

The antiepileptic evaluation was carried out using maximal electro shock induced seizure method. Swiss strain albino mice of either sex weighing 25-30 g were used for this study. The test compounds were suspended in 0.5 % v/v Tween 80 in water. LD50 of the newly synthesized compounds were determined by Miller and Tainter [50] method by administering the compounds intraperitoneally.

2.11.1. Acetic acid induced writhing method [51]

The animals were housed and acclimated under standard laboratory conditions and were supplied with food and water ad libitum. The animals were divided into ten groups of six mice each. The control group of animals was administered with 0.5% v/v Tween 80 (0.5 ml) suspension. The standard drug was administered with paracetamol (Micro Labs) i.p. in a dose of 2.5 mg kg-1. Tween 80 suspension (0.5 % v/v) of the test compounds were administered i.p. in a dose of 20 mg.kg-1. After 20 min. of the administration of the test compounds and standard, all the groups of mice were given the writhing agent 3 % v/v aqueous acetic acid in a dose of 2 ml kg-1 i.p. The total number of writhing produced in these animals were counted visually for 15 min. and the number of writhing produced in treated groups were compared with those in control group. The results recorded in Table 3 are expressed as percentage protection and are analyzed statistically by “student t test”.

2.11.2. Anti-inflammatory activity

The antiinflammatory activity was evaluated by carrageenan induced paw edema method [52]. Albino rats of Wistar strain weighing 100-200 g of either sex were divided into ten groups each of six animals. Tween 80 suspension (0.5 % v/v) of the test compounds were administered intraperitoneally in a dose of 20 mgkg-1. The control group was given only 0.5% v/v Tween 80 (0.5 ml) suspension. One group was administered with diclofenac sodium (Novartis Laboratories) as standard, i.p. in a dose of 2 mg kg-1. After 30 min. of the administration of test compounds paw edema was induced in albino rats by injecting 0.1 ml of carrageenan (1% v/v in normal saline) suspension, into subplantar region of the left hind paw of each rat. After 1, 2 and 3 h of carrageenan injection, the increase in paw volume was measured by a plethysmometer. The antiinflammatory activity was measured in terms of percentage inhibition of edema and is analyzed statistically by “students t test” and recorded in Table 4.

The antiepileptic activity was evaluated by maximal electroshock induced seizure (MES) method. Swiss albino mice weighing 25-30 g of either sex were divided into ten groups each of six animals. The control group of animals was administered with 0.5%v/v Tween 80 (0.5 ml) suspension. One group was administered with phenobarbitone (Nicholas Primal) as standard, i.p. in a dose of 20 mg.kg-1. Tween 80 suspensions (0.5 % v/v) of the test compounds were administered i.p. in a dose of 20 mg kg-1 for other group of animals. After 1 h of the administration of the test compounds and standard, the animals were given electroshock through ear electrodes of 150 mA for 0.2s by electroconvulsiometer. Onset times for tonic, flexion, extension and clonic phase were noted. The protective index was observed as reduction time of tonic extensor phase and was tabulated in Table 5.

Minimal motor impairment was measured in mice by the Rota rod (Techno, India) test. The mice were trained to stay on an accelerating Rota rod of diameter 3.2 cm that rotates at 10 revolutions per min. Previously trained Albino mice were given test compounds i.p. in doses of 20, 50, 100, and 200 mg.kg-1. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least one min. in each of the three trials. The results are summarized in Table 1.

2.11.5. Behavioral test

The titled compounds (20 mg kg-1 in 0.5 % v/v Tween 80 suspension) were screened for their behavioral effects using actophotometer (Techno, India) by Boissier and Simon method [54]. Albino mice were placed inside the actophotometer after 30 min. of administration of test compounds i.p. The behavior of animals inside the photocell was recorded as a digital score. The control animals were administered with 0.5 ml of Tween 80 (0.5% v/v) suspension. The observations are summarized in Table 2.

Infrared spectrum of all eight compounds showed strong absorption bands for C=N group at 1393 cm-1 and aromatic absorptions at 1609 cm-1 and below 900 cm-1. Compound 2 showed strong absorptions for SO2 group at 1151 cm-1 and N-H group at 3380 cm-1. Compound 4 showed strong absorptions for NO2 group at 1508 cm-1. Compound 5 showed strong absorptions for OH group at 3424 cm-1. Compound 6 showed absorption band for NH group at 3434 cm-1. Compound 7 showed absorption band for N-H at 3434 cm-1 and for NO2 group at 1552 cm-1. Compound 8 showed absorption band for N-H group at 3428 cm-1. Compound 5 showed strong absorptions for OH group at 3432 cm-1 and for sulphonic acid group at 1116 cm-1.

Minimum motor impairment was measured in mouse by Rota rod test showed that all the synthesized compounds have no effect which was indicated by their ability to maintain the equilibrium on the Rota rod for more than 1 min.

3.2.2. Behavioral test

Compound 7 produced significant decrease in the spontaneous motor activity in mice. This effect was dose dependent and the effect was observed within 30 min. of drug administration. Other compounds except compound 3 also showed good behavioral activity.

3.2.3. Anti-nociceptive activity

3.2.3.1. Acetic acid induced writhing method

Antinociceptive activity was evaluated by acetic acid induced writhing method. All the compounds tested exhibited antinociceptive activity in a dose of 20 mg.kg-1. The antinoci-ceptive activity of compounds 4, 7 and 8 is found to be superior compared to other synthesized compounds. The compounds 2, 3 and 5 exhibited moderate antinociceptive activity. The compounds 6 and 9 exhibited negligible anti-nociceptive activity.